Voltage gated calcium channels (VGCC or calcium channel) mediate Ca2+ influx in excitable cells. Upon depolarization of the plasma membrane, calcium channels undergo a series of conformational changes that begin with charge movement resulting in the opening of a pore or conductance pathway that is selective for the influx of calcium ions (Catterall, W. A. (1988) Science 242:50-61 and Bean B P. (1989) Annu. Rev. Physiol. 51:367-368).
Calcium channels are a diverse class of proteins that have been traditionally separated into at least six different types based on their electrophysiological and pharmacological properties. The groups are referred to as L-type (for Long Lasting), T-type (for Transient), N-type (for neither L nor T, or for “Neuronal”), P-type (for Purkinje cell), Q-type and R-type (for resistant) Hess, (1990), Ann. NY Acad. Sci. 560:27-38; Bertolino and Llinás, (1992) Annu. Rev. Pharmacol. Toxicol. 32:399-421; Randall and Tsien, (1995) J. Neurosci. 15:2995-3012). Except for the T type calcium channel, which is low voltage activated (LVA), the L-, N-, P-, Q- and R-types are all high voltage activated (HVA), i.e. their activation thresholds are normally above −40 mV.
The best characterized calcium channel is the rabbit skeletal muscle dihydropyridine (DHP)-sensitive L-type calcium channel. It is composed of four tightly associated subunits α1, α2δ, β and γ (Catterall et al, supra, Hosey, et al. (1989) Ann. N.Y. Acad. Sci. 560:27-38). All of these subunits from rabbit have been characterized by molecular cloning (Tanabe et al, (1987) Nature 328:313-318; Ellis et al, (1988) Science 241:1661-4; Ruth et al, (1989) Science 245: 1115-1118; and Jay et al, (1990) Science 248:490-492).
The neuronal conotoxin GVIA sensitive N-type calcium channel has also been purified (Witcher et al, (1993) Science 26:486-9). It contains α1B, α2δ, and β subunits but lacks a skeletal muscle-like γ subunit. The α1 subunit cDNA clones of skeletal muscle L- and neuronal N-type calcium channel are termed α1S and α1B, respectively. It has been shown that the α1 subunit alone can form a functional channel (Perez-Reyes et al, (1989) Nature 340:233-6 and Tanabe et al, (1988) Nature 336:134-139) while the α2δ and β subunits play a regulatory role (Lacerda et al, (1991) Nature 352:527-530; Birnbaumer et al, (1998) J. of Bioenergetics and Biomembranes 30:357-375; and Qin et al, (1998) Am. J. Physiol. (Cell Physiol.), 274: C1324-C1331). The α2δ and β subunits regulate almost all aspects of the channel properties including tight coupling between charge movement and pore opening, voltage dependent activation and inactivation, and prepulse potentiation.
To date, six non-allelic genes have been cloned encoding neuronal HVA-calcium channel α1 subunits (referred to as α1A through α1F, and α1S) (Mikami et al, (1989) Nature 340:230-233; Snutch et al, (1990) PNAS (USA) 87;3391-395; Mori et al, (1991) Nature 350:398-402; Hui et al, (1991) Neuron 7:3-44; and Williams, (1992) Science 257:389-395) and three have been cloned encoding LVA-calcium channel α1 subunits (α1G-α1I) (Perez-Reyes et al, (1998) Nature 391:896-900 and Lee et al, (1999) J. Neurosci. 19:1912-1921). Analyses of these sequences indicate that the primary sequences of the calcium channel cDNAs have homologies ranging from between 40%-70%. Hydropathicity analyses indicate that, like voltage-dependent sodium channels, calcium channel α1 subunits contain four homologous repeat transmembrane domains (domain I through IV). Each of these four domains contains five hydrophobic putative transmembrane spanning helices, referred to as S1-S3, S5 and S6, and one amphipathic segment (S4). The amphipathic segment contains highly-conserved, positively-charged amino acids every 3rd or 4th residue and this segment is thought to serve as the voltage sensor of the channel. The α1 subunit determines the functionality of the Ca2+ channel (α1C, α1D and α1F for L-type, α1A for P/Q-type, α1B for N-type, α1E most likely for R-type, and α1G to α1I for T-type).
Molecular cloning of calcium channels has also revealed that there are seven different types of α1 subunits for the (HVA) calcium channel, three types of α2δ subunits (Ellis et al, supra; Lugbauer et al, (1999) J. Neurosci. 19:684-691) and four types of β subunits (Ruth et al, supra; Pragnell et al, (1991) FEBS Lett. 291:253-258; Perez-Reyes et al, (1992) J. Biol. Chem. 267:1792-1797) and Castellano et al, (1993) J. Biol. Chem 268:12359-12366 and Castellano et al. (1993) J. Biol. Chem 268: 3450-3455).
Recently, the analyses of Drosophila genomic sequences have revealed that there are four different types of α1, three types of α2δ and one type of β subunit in Drosophila genome (Littleton and Ganetzhy, (2000) Neuron 26:35-43). Ten different mammalian α1 subunits have been identified. Based on the ratio of α1 and α2δ subunits (4 to 3) in Drosophila, there should be more than three types of α2δ subunits in mammals. No regulatory subunits of T-type channels (α1G to α1H) have been identified yet, further suggesting that there are more VSCC subunits to be identified.
Understanding the molecular properties of the mature calcium channel subunits, their precursor proteins and the regulation of the calcium channel subunits require identification of a variety of calcium channel subunit nucleic acid sequences. An understanding of calcium channel subunit gene regulation is important for the identification of therapeutic agents affecting calcium channel function. Furthermore, the identification of a variety of nucleic acid sequences coding for calcium channel subunits is needed for the diagnosis of gene defects associated with calcium channel-implicated diseases.
A number of compounds useful in treating various diseases are thought to exert their beneficial effects by modulating voltage dependent calcium channel function. Many of these compounds bind to calcium channels and block or reduce the rate of Ca2+ influx into cells in response to membrane depolarization. An understanding of the pharmacology of compounds that interact with calcium channels and the design of such compounds is limited by an understanding of the genes that code for them. Moreover, the identification of calcium channel subunits is needed to recombinantly produce sufficient quantities of highly purified channel subunits. With the availability of large amounts of purified calcium channel subunits, functional channels can be prepared and used in screening assays to identify or determine the effect of various compounds on channel function thereby providing a basis for the design of therapeutic agents which affect the calcium channel. Thus there is a need to further study the structure, subunit interaction, and channel composition of calcium channels.
A calcium channel α2δ subunit has been identified in every voltage-dependent calcium channel purified to date from various mammalian tissues including rabbit skeletal muscle and rabbit brain. Structurally, the α2δ subunit is a heavily glycosylated protein dimer that is encoded by a single gene and post-translationally cleaved to yield α2δ and δ subunits linked by a disulfide bond. Experimental evidence suggests a single transmembrane topology located in the δ subunit of the α2δ subunit (Gurnett, et al (1996) Neuron 16:431-40; Gurnett et al. (1996) J. Biol. Chem. 271:27975-8, and Felix, et al. (1997) J. Neurosci. 7:6884-910). The α2δ subunit regulates most of the properties of the calcium channels, including voltage dependent kinetics and ligand binding (Qin et al, supra).
Characterizing the effects of the calcium channel subunit on ligand binding demonstrated that the α2δ subunit alters the binding of neurological and cardiovascular drugs to the ion channel pore-forming α1 subunit. Recently, gabapentin, a novel anticonvulsant drug, was shown to bind with high affinity directly to the calcium channel α2δ subunit (Gee, et al. (1996) J. Biol. Chem. 271:5768-76). Gabapentin may control neuronal excitability by modifing calcium channel activity or expression (Rock et al, (1993) Epilepsy Res. 16:89-98). More interestingly, antibodies directed against the α2δ subunit block secretion from PC12 cells, suggesting that the α2δ subunit may play a distinct role in neurotransmitter release (Gilad et al (1995) Neurosci. Lett. 193:157-60; Tokumaru, et al. (1995) J. Neurochem. 65:831-836 and Wiser, et al. (1996) FEBS Lett. 379:15-20).
Ca2+ ions play very important roles in normal cellular function including neurotransmitter release, cellular signaling, smooth and skeletal muscle contraction and gene expression. Regulation of intracellular Ca2+ level is at the center of multiple systems for controlling numerous cellular functions. An abnormal intracellular Ca2+ level is implicated in diseases such as neuropathic and chronic pain, migraine, Lambert-Eaton Syndrome, anxiety, seizures, epilepsy, ischemia, trauma, stroke Schizophrenia and Alzheimer's Disease as well as many other types of neuronal degeneration. Elevated or dysregulated Ca2+ is also important in neuronal plasticity.
The defective α2δ gene has also been associated with proliferative diseases such as cancer and inflammation. Treatment with compounds that bind to α2δ to changes in the signal transduction mechanism of certain proteins including altered levels of MEK (MAP kinase kinase), an enzyme that activates the MAP kinase (mitogen-activated protein kinase). Inhibitors of MEK appear to mimic the analgesic activities associated with the binding of gabapentin to α2δ. Activation of MAP kinase by mitogens appears to be essential for proliferation and constitutive activation of this kinase is sufficient to induce cellular transformation.